Polymixin b

The selected clones were sequenced, and plasmids extracted from these clones were used for transformation of HB2151 cells. A single and freshly transformed colony was added to 3 ml 2YT medium with 100 µg/ml ampicillin and 2% (w/v) glucose, incubated at 37°C with vigorous shaking at 250 rpm for 3–4 h, and then transferred into 200 ml of SB medium with 100 µg/ml ampicillin for continued incubation until optical density of the culture at 600 nm reached 0.6. Next, IPTG (isopropyl-1-thio-β-d-galactopyranoside) was added to a final concentration of 1 mM to induce protein expression, and the culture was further incubated overnight at 30°C, 250 rpm. Bacteria were collected by centrifugation at 8,000 rpm for 10 min and re-suspended in 30 ml PBS buffer. Polymixin B (Sigma-Aldrich) (0.5 µm/ml) was added to the bacteria solution (1:1,000). After 30 min incubation with rotation at 250 rpm at 30°C, the suspension was centrifuged at 8,000 rpm for 10 min at 4°C. Proteins were purified using protein G column (Roche Applied Science) according to the manufacturer’s protocol. The degree of protein purity was determined by SDS-PAGE, and protein concentration was measured spectrophotometrically.

Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by density gradient centrifugation using Ficoll-Pague Plus (GE Healthcare). PBMCs were cultured in RPMI 1640 supplemented with 5% (v/v) fetal calf serum (Gibco), 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin at a density of 1 to 1.5×10⁶ cells per well in 48-well plates. Cells were stimulated with 1 µg/mL phytohemagglutinin M (Calbiochem) in the presence or absence of different HDL preparations and incubated for 20 hours at 37°C in a CO₂ incubator. To prevent potential effects of endotoxin contamination, cell culture medium was supplemented with 1 μg/mL polymixin B (Sigma-Aldrich); HDL and phytohemagglutinin M were preincubated with polymyxin B for 15 minutes before being added to PBMC. Tumor necrosis factor-α, interleukin-1β (IL-1β), IL-6, and macrophage inflammatory protein-1β were measured in cell-free supernatants using Human Cytokine Magnetic 4-Plex Panel (R&D Systems).

Human and canine DCs were generated using a three-step protocol as described previously [23 (link)]. First, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using standard procedures (Ficoll-Paque Plus^®; GE Healthcare, Uppsala, Sweden). Second, monocytes were purified from PBMCs by magnetic cell sorting using an anti-CD14 monoclonal antibody conjugated to magnetic beads (MACS^®; Miltenyi Biotec, Bergisch Gladbach, Germany). Third, DCs were generated by culturing CD14⁺ monocytes at 1 × 10⁶ cells/mL for 6 days in RPMI-1640 (Life Technologies, Gaithersburg, MD, USA), supplemented with 10% foetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM l-glutamine (Gibco Invitrogen Corp., Grand Island, NY, USA), 100 U/mL polymixin B (Sigma-Aldrich, St. Louis, MO, USA), and specific recombinant human (rh) or canine (rc) differentiation factors. In particular, canine monocyte cultures were supplemented with 40 ng/mL rhGM-CSF and 30 ng/mL rcIL-4 (R&D Systems Inc., Minneapolis, MN, USA), while human monocyte cultures were supplemented with 20 ng/mL rhGM-CSF and rhIL-4 (R&D Systems Inc.) every 2 days.

Stx expression by the STEC strains was determined according to the method adapted from Shringi et al. [28 (link)] using an ELISA kit (Premier EHEC, Meridian Bioscience, Ohio, USA). Mitomycin C (Sigma Aldrich, Missouri, USA) was used at a final concentration of 0.5 μg/mL to induce Stx production. After induction, the cells were lysed using Polymixin B (Sigma Aldrich, Missouri, USA) at a final concentration of 0.5 mg/mL and incubated at 37°C for 1 h with rotary shaking (250 rpm). Polymixin B treated cultures were diluted 1 : 100 in sterile LB broth immediately followed by 1 : 2 dilution in sample diluent of the ELISA kit. Absorbance readings were obtained at wavelengths 450 nm and 630 nm using a Victor X microtiter plate reader (Perkin Elmer, Glen Waverley, Australia) and the results were displayed as the mean value of two independent biological replicates.

Following a previous study, hippocampal administration of S1 protein was performed^{27 (link)}. Eight-week-old mice were used for the spike protein injections. S1 protein was purchased from Acrobiosystems (Cat #S1N-C52H4, Newark, DE, USA), and used after polymixin B (Cat #P1004, Sigma-Aldrich, St. Louis, MO, USA) treatment (30 µg/ml). The animals were anesthetized with isoflurane, and the injection paths were drilled into their skulls. Then, 5 µg of S1 protein (1 µg/µl) were bilaterally injected into each hippocampal region using a Hamilton syringe (Cat #80330, Hamilton Company, Reno, NV, USA) attached to a syringe pump (Cat #53311, Stoelting Co., Wood Dale, IL, USA) at a constant volume of 0.5 µl/min. The injection coordinates of the hippocampus were 1.5 (ML), − 2.06 (AP), and − 2.0 (DV) from the bregma.

Overnight P. aeruginosa PA14 or E. coli lptD4213 cultures were diluted 1:100 and grown to mid exponential phase at 37 °C. Cultures were diluted 1:10 into PBS and treated with antibiotics for 15 min. P. aeruginosa PA14 was stained with TO-PRO-3 (640 nm excitation, 670/30 nm emission) to measure cell membrane integrity. E. coli lptD4213 was stained with both TO-PRO-3 and DiOC2(3) (ThermoFisher B34950) to measure cell membrane integrity and membrane potential. DiOC2(3) was evaluated as a ratio of green (488 nm excitation, 525/50 nm emission) to red (488 nm excitation, 610/20 nm emission)^{66 (link)}. The LSRII flow cytometer (BD Biosciences) at the Flow Cytometry Resource Facility, Princeton University, was used to measure the fluorescent intensities of both dyes in response to antibiotic treatment. 100,000 events were recorded for each data file. Gates for permeabilization were determined using polymixin B (Sigma-Aldrich P1004) and untreated controls. Gates for depolarization were determined using carbonyl cyanide m-chlorophenyl hydrazone (CCCP) as a positive control. Data were analysed using FlowJo v.10 software.

Four days after C57BL/6J mice were injected intraperitoneally with 2 mL of 4.05% BBL™ Brewer modified thioglycolate medium (Becton Dickinson, Sparks, MD, USA), their peritoneal macrophages were collected with two peritoneal lavages of 5 mL of cold PBS. The harvested cells were centrifuged at 1,300 × g for 10 min and suspended in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) containing 8% FBS. Red blood cells were removed with lysis buffer (0.83% NH₄Cl, 0.01 M Tris-HCl [pH 7.2]) and then washed away with medium. The macrophage suspension was added to a 96-well microplate at 3 × 10⁵ cells/well and incubated at 37°C in a 5% CO₂ incubator for 4 h, allowing the cells to settle to the bottom. The wells were washed with FBS-free DMEM to remove the floating cells, and incubated for 20 h after the addition of the indicated stimulants, including positive and negative controls. To confirm the effects of the resident lipopolysaccharide (LPS) in the protein samples, polymixin B (Sigma) was used.